Cancer Immunotherapy Targeting Survivin Commentary Re: V

6310 Vol. 9, 6310–6315, December 15, 2003 Clinical Cancer Research

The Biology Behind Cancer Immunotherapy Targeting Survivin Commentary re: V. Pisarev et al., Full-Length Dominant-Negative Survivin for Cancer Immunotherapy. Clin. Cancer Res., 9: 6523–6533, 2003.

John C. Reed1 and Darcy B. Wilson2 human tumor cell lines using antisense methods, or interfering 1The Burnham Institute and 2The Torrey Pines Institute for with the function of the endogenous Survivin protein by ectopic Molecular Studies, La Jolla, California expression of dominant-negative Survivin mutants, results in arrested cell division and apoptosis, implying that tumor cells cannot replicate and survive without this important and nonre- The Survivin protein regulates both cell division and cell dundant protein. Consequently, it stands to reason that tumors survival and is overexpressed in the vast majority of human could not easily escape therapeutic interventions targeting Sur- cancers (1). In fact, the gene encoding Survivin is notable for its vivin by simply shutting-off its expression. high degree of tumor-specific expression, ranking among the top With these issues in mind, the Gabrilovich group set out to five most tumor-specific genes in the human genome based on ask whether peptides-derived from the Survivin protein could comparisons of the number of times survivin transcripts appear stimulate cytolytic T-cell responses in vitro, using peripheral in tumors compared with normal cells and tissues (reviewed in blood lymphocytes (PBLs) derived from normal volunteers and Ref. 2). In normal cells, Survivin is produced only in small prostate cancer patients. The strategy they used involved infect- amounts and only briefly during mitosis. However, in tumors, the Survivin protein is continuously present at excess levels, ing autologous dendritic cells (DCs) with recombinant adeno- suppressing apoptosis and aiding in cell division (reviewed in virus expressing the full-length Survivin protein in the hope that Ref. 3). Consequently, Survivin has emerged as a hot target for at least some of the endogenously processed peptides from cancer therapy, with a variety of strategies already articulated Survivin could be displayed in immunogenic forms on HLA-A2, for nullifying Survivin in tumors, including (a) small-molecule the most common of the HLA class I antigens expressed in antagonists that block interactions of Survivin with critical Caucasian populations. To avoid any potential pro-oncogenic partner proteins (4–6); (b) antisense oligonucleotides that re- side effects, a dominant-negative mutant of Survivin [containing duce survivin expression; (c) ribozyme-mediated inhibition of a single amino acid substitution (T34A)] was used instead of the survivin expression (7); and (d) gene therapy using dominant- wild-type protein for in vitro immunizations (8). negative mutants of Survivin that induce cell cycle arrest and Elegant studies of the mechanisms of antigen processing apoptosis (8). In this issue of the Clinical Cancer Research, the and presentation have revealed that immunogenic peptides of Gabrilovich group [Pisarev et al. (9)] describe preclinical stud- 8–9 amino acids in length are displayed on cell surface HLA ies aimed at yet another strategy for exploiting Survivin as a class I molecules after degradation of intracellular proteins via cancer target, namely, using survivin to create a tumor vaccine. specialized proteasomes, and transport of the resultant peptides Inducing cell-specific immunity against tumors represents to the endoplasmic reticulum where they associate with class I a long-standing goal of cancer immunology (10). To be suc- antigens and then are transported to the plasma membrane (Ref. cessful, several conditions must be met, including identifying 12; Fig. 1). Thus, the 142-amino-acid human survivin protein tumor-associated antigens (TAAs) that are capable of being could theoretically generate 136 nonamer peptides. Rather than presented on human leukocyte class I or II antigens (HLA-I or testing all those possible peptide sequences to see whether they -II) and stimulating host T cells using the available repertoire of are reflected in the repertoire of T-cell responses made to T-cell antigen receptors (reviewed in Ref. 10). Ideally, such full-length Survivin, the Gabrilovich group took some educated TAAs should be displayed only on tumor cells and not normal guesses. Using data from the literature (13, 14) and predictive cells, or, at least, such antigens should be expressed only on computer algorithms, they surmised that three peptide sequences nonessential normal tissues if tumor specificity is impossible contained within Survivin ought to be generated in antigen- (e.g., breast, prostate, ovary, testes). Furthermore, the TAA presenting cells and should be capable of binding HLA-A2. The should be an essential gene, so that tumors cannot avoid immune strategy thus involved in vitro stimulation of autologous PBLs surveillance simply by refusing to express it. with Survivin-adenovirus-infected DCs, followed by testing of Gene ablation studies in mice have confirmed that survivin the resulting activated T cells for their ability to recognize is an essential gene (11). Knocking-down survivin expression in HLA-A2-expressing cells pulsed with one of the three synthetic Survivin 9-mer peptides. The question asked was whether T cells could be enticed, at least in vitro, to respond to a self- antigen (Survivin), thus breaking tolerance. Secondary ques- Received 10/31/03; accepted 11/4/03. tions involved addressing the issues of whether such Survivin- Grant support: NIH CA-78040 and AG15402. specific T cells could kill either peptide-pulsed target cells or a Requests for reprints: John C. Reed, The Burnham Institute, 10901 tumor cell line that endogenously expresses Survivin and HLA- North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 646-3132; A2, and whether normal hematopoietic stem cells would be Fax: (858) 646-3194; E-mail: [email protected] or Darcy B. Wilson, Torrey Pines Institute for Molecular Studies, 3550 General affected inasmuch as they are among the nontransformed types Atomics Court, La Jolla, CA 92121-1122. Phone: (858) 455-3840; of cells known to express at least low levels of Survivin in vivo Fax:(858) 455-3804; E-mail: [email protected]. (15). Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. Clinical Cancer Research 6311

Fig. 1 Antigen processing and presentation. Intracellular proteins are proteolysed by proteosomes in the cytosol, and the resulting peptides are transported into the lumen of the endoplasmic reticulum via the TAP transporter. Peptides associate with HLA (human) or MHC (mouse) class-I antigens, which are transported eventually to the cell surface.

PBLs derived from all four normal subjects and the four T-cell lines were tested on peptide-pulsed DCs, using the fre- cancer patients resulted in T-cell populations capable of re- quency of lymphokine-producing cells as a measure of response sponding to at least one of the three synthetic Survivin peptides, (“ELISPOT assay”), five of six HLA-A2-positive healthy do- after three rounds of successive stimulation with Survivin-ade- nors and all four HLA-A2-positive cancer patients showed only novirus-infected DCs. Moreover, seven of the eight PBL spec- an approximate doubling in the number of Survivin-responsive imens responded to two of the three Survivin peptides. Thus, T cells, compared with background controls. Similarly, cytolytic these findings argue that, at least in vitro, it is possible to break killing of peptide-pulsed cells by the stimulated T cells was tolerance and encourage T cells within the available repertoire relatively feeble, with only a ϳ10% increase in specific lysis, to respond to Survivin peptides in the context of HLA-A2. sometimes requiring high E:T ratios of 100:1. Of course, one of Although an encouraging observation, these findings only begin the limitations of these experiments is that the peptides chosen to scratch the surface and many questions remain to be ad- to measure responsiveness to Survivin are only guesses of what dressed toward the goal of establishing whether Survivin-based the T-cell repertoire might produce. Of the 136 theoretically tumor vaccines are likely to be safe and effective in cancer possible 9Јmer peptides represented in the human Survivin patients. protein sequence, only 3 were tested. On the basis of the First, regarding efficacy, from the information provided, it SYFPEITHI algorithm, which assesses peptide sequences for is unclear how efficiently Survivin-transduced DCs activated T HLA binding,3 probably at least five peptides in the Survivin cells within the available repertoire of PBLs of normal subjects sequence are candidate HLA-A2-binders. Thus, the actual fre- and prostate cancer patients. One would like to know some of quency of Survivin-responsive T cells in the normally HLA the details about the frequency of responding T cells in periph- heterozygous human population might be significantly higher eral blood and whether that frequency changes in cancer pa- than estimated from best-guess approaches, speaking to the need tients, to make comparisons of the merits of Survivin relative to for a more systematic survey of the possible “peptide space” in other TAAs that might have been used as an alternative (e.g., prostate-specific antigen; prostate-specific membrane antigen for prostate cancer or telomerase for many types of cancer). According to the data shown, when the Survivin-stimulated 3 Internet address: http://syfpeithi.bmi-heidelberg.com.

Fig. 2 Apoptosis pathways and Survivin. Apoptosis pathways are presented, showing some of the routes of caspase activation. Left, granzyme B (GraB; purple) injected into target cells by natural killer (NK) cells and CTLs can directly cleave and activate several caspases. Middle, tumor necrosis factor (TNF)-family death receptors such as Fas and TRAIL receptors recruit adapter protein FADD to their cytosolic domains, which in turn binds caspases-8 (casp-8) and (in humans) -10. Right, mitochondria release cytochrome c (cyto-C) into the cytosol, which binds Apaf1, resulting in caspase-9 (casp-9) activation. Upstream initiator caspases [e.g., caspases-8 (casp-8), -9 (casp-9), and -10] cleave and activate downstream effector caspases (e.g., caspases-3, -6, and -7), which then cleave substrate targets, including polyADP ribosylpolymerase (PARP) and ICAD/DFF45, as well as many others not depicted in the figure. Bid acts as one of the points of cross-talk between the GraB and death receptor pathways and the mitochondrial apoptosis pathway, whereby this pro-apoptotic Bcl-2-family member is activated on cleavage by either GraB or caspase-8. Activated Bid (BID) translocates to mitochondria and promotes cytochrome c release. Some of the major apoptosis suppressors known to be overexpressed in

cancers are shown, including (a) FLIP, which binds pro-caspases-8 and -10, interfering with their activation; (b) Bcl-2 and Bcl-XL, which insert in mitochondrial membranes and block cytochrome c release; (c) XIAP, which directly binds and inhibits the activated forms of caspases-3, -7, and -9; and (d) Survivin, which appears to bind pro-caspase-9, preventing it from becoming activated by Apaf1s/cytochrome c.

which the use of Survivin as a TAA is concerned. It should be the lumen of the endoplasmic reticulum, as well as loss of emphasized that only T-cell responses directed toward antigens expression of the HLA antigens required for presenting TAAs to displayed on HLA-A2 were examined, and, therefore, the con- T cells (reviewed in Refs. 16 and 17). The study by Pisarev et tributions of other HLA-A allele products and members of the al. used, in part, PBLs from prostate cancer patients. Advanced other HLA families (e.g., HLA-B and HLA-C) remain to be prostate cancers are known for their loss of expression of defined. HLA-A antigens, whereas HLA-B and HLA-C seem to be more Indeed, an issue to consider in any tumor vaccine strategy commonly preserved (18). Thus, it might be necessary to exploit that relies on cell-mediated immunity is the question of whether a combination of HLA antigens for presentation of survivin- the target tumor cells are competent to process and express derived peptides to ensure adequate T-cell stimulation. An ideal antigen on class I antigens. In this regard, mechanisms of tumor TAA for vaccines, therefore, ought to be capable of generating resistance to CTLs can include reduced expression or activity of several endogenous peptides that can be effectively presented in the proteins responsible for digesting whole tumor-associated the context of various different HLA antigens. Thus far, only proteins into small peptide antigens and transporting them into responses in the context of HLA-A2 have been explored.

Fig. 3 Afferent and efferent limbs of cell-mediated immune response. The afferent and efferent limbs of the cellular immune response to tumors are depicted. T cells recognizing tumor-associated antigens (TAAs) displayed on class I antigens on tumors clonally expand (AFFERENT), then differentiate into cytolytic killers and induce apoptosis of the tumor target cells (EFFERENT).

Thus, although it is unlikely that prostate cancers or other vivin using T cells derived from cancer patients, as opposed types of tumors will completely lose expression of all types to the normal donors used, asking whether cancer patients of HLA-class I antigens because of ensuring natural killer can mount a response, and also using autologous tumor cell (NK) cell-mediated attack that would result (19), a successful lines or at least tumor cell lines derived from the same tissue tumor vaccine might require that more than one type of HLA of origin (in this case, prostate) as the patients’ cancer. antigen be capable of displaying immunogenic peptides that Second, regarding safety, most likely, Survivin is ex- break tolerance to self. pressed in all types of normal cells during division, but only Also at issue with respect to efficacy is the question of transiently and only at low levels, compared with tumor cells. whether the Survivin-stimulated T cells can respond robustly Although Pisarev et al. have attempted to address the issue of to tumor cells expressing endogenous Survivin (as opposed to T-cell reactivity toward normal hematopoietic CD34ϩ stem peptide-pulsed cells). Thus far, the Gabrilovich group has cells, finding essentially none, this initial foray into the examined only one human tumor cell line that intrinsically subject does not exclude the possibility of autoimmune reac- expresses elevated levels of Survivin and that displays tions against other types of Survivin-expressing cells such as HLA-A2 (e.g., MCF7 breast cancer cell line), using cell competition experiments to indirectly infer a response to those seen in the gut, skin, and other tissues in which cell endogenous Survivin in the context of HLA-A2. To defini- replication occurs. Even activated lymphocytes express en- tively demonstrate a response to endogenous Survivin, one dogenous Survivin when stimulated to proliferate (20), rais- would need to knock-down Survivin expression using small ing the possibility of inducing populations of Survivin- interfering RNA (siRNA), antisense, or some other method, responsive T cells that attack themselves in an autoantigen showing that T cells fail to lyse tumor cells when the putative response that could be detrimental to immune-defense and TAA is no longer made. Alternatively, specifically neutral- counterproductive to mounting an effective antitumor re- izing HLA class I antigens with an antibody might at least sponse. Clearly, extending the preclinical studies to animal indicate whether the lysis observed is HLA-restricted, as models is necessary to gain a better understanding of the opposed to occurring through the non-class-I-restricted relative risks of efforts to exploit Survivin as a TAA. mechanisms typically seen with lymphokine-activated killer Finally, it is interesting to contemplate anticancer strategies and NK cells that are likely to be present in the same cell targeting Survivin with respect to its dual role as a possible TAA preparations. Finally, it would be interesting to contrast the versus its function as an antiapoptotic protein. Although most lytic activity against tumor cells expressing endogenous Sur- efforts to develop tumor vaccines focus on breaking tolerance

and triggering tumor recognition by T cells, scant attention is Acknowledgments generally given to the question of whether the responding T We thank Judie Valois for manuscript preparation, Tom Kipps for cells can effectively kill the tumor target cells once recognized. helpful discussions and suggestions, and the NIH for generous support. The cytolytic mechanism induced by T cells involves activation of specific apoptosis pathways, involving caspase-family References cell death proteases (21). Survivin is a member of the inhib- itors of apoptosis protein (IAP)-family of apoptosis suppres- 1. Altieri, D. C., Marchisio, P. C., and Marchisio, P. C. Survivin apoptosis: an interloper between cell death and cell proliferation in sors, a group of antiapoptotic proteins known for their ability cancer. Lab. Investig., 79: 1327–1333, 1999. to directly bind and suppress caspases (reviewed in Ref. (22). 2. Reed, J. C. The survivin saga goes in vivo. J. Clin. 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Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. Cancer Immunotherapy Targeting Survivin: Commentary re: V. Pisarev et al., Full-Length Dominant-Negative Survivin for Cancer Immunotherapy. Clin. Cancer Res., 9:6523−6533, 2003.

John C. Reed and Darcy B. Wilson

Clin Cancer Res 2003;9:6310-6315.

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